Cancer gene therapy would benefit greatly from the availability of a vector that has a high efficiency of gene expression and the ability to target tumors. A number of transfection systems have been developed to deliver heterologous genes into tumors in vivo to investigate cancer gene therapy, but all have limitations. For example, retroviral vectors have been used for gene delivery because they mediate stable gene transfer with a low potential for immunogenicity; however, transfer efficiencies are relatively low (1-4) and germ line modification is a potential problem (5). In addition, retroviral vectors, with few exceptions, are susceptible to lysis by serum components in human blood (6-8). This greatly limits their in vivo applications. Adenoviral vectors appear to be more efficient for gene transfer in vivo, but these vectors may cause toxicity to patients due to the highly immunogenic properties of adenoviral proteins (9, 10). Additionally, they are commonly used in a localized manner because they generally lack the ability to be delivered via the bloodstream (11-13). In fact, they may cause severe toxicity if administered intravenously (77).
An alternative viral vector system that has numerous advantages is based on Sindbis virus. Sindbis virus has been studied extensively since its discovery in Egypt in 1953 (14-16, 69). Gene transduction based on Sindbis virus, a member of the alphavirus genus, has been well-studied (17-27). Sindbis vectors show extremely high efficiency of gene transfer. They are plus-strand RNA viruses, which through a process of amplification in the cytoplasm of infected cells can express 105 active RNA species per cell within a few hours after infection. This level of RNA amplification allows for very high levels of expression of the transferred gene product, which would allow for prolonged expression were it not for the apoptotic nature of the virus (28-31).
Based on recommendations for the handling of alphaviruses and other arboviruses in the laboratory (32), Sindbis virus is considered fairly safe. Sindbis virus is endemic to many parts of the world where it is associated with minimal, transient disease (17, 33, and 34). Whereas the majority of alphaviruses require Level 3 practice and containment and/or vaccination, Sindbis requires only Level 2. Level 2 practice and containment is assigned to viruses whose infection results either in no disease or in disease that is self-limited (32, 33, 34). Replication incompetent Sindbis vectors derived from Sindbis viruses, such as those referred to herein, can be considered even safer as their capacity to infect and replicate to cause viremia or disease is virtually non-existent. It has been stated that they can be considered safe enough for level 1 use (ACGM Compendium Of Guidance. Guidance From The Health And Safety Commission's Advisory Committee On Genetic Modification. Part 2—Annex III—Guidance On Commonly Used Viral Vectors, issued March 2000. Available on the Worldwide Web at hse.gov.uk. The capacity to infect and replicate to cause viremia can only be reacquired through recombination, which can be minimized and monitored.
Sindbis vectors also avoid potential complications associated with chromosomal integration (27). Recent methods have added substantial ease to engineering new Sindbis vector constructs capable of nonreplicative infection and further enhanced safety aspects of the vector (17). Because Sindbis virus is a blood-borne virus (33) and can cross the blood brain barrier (18), vectors based on this virus are among the few available ones that are capable of migrating through the bloodstream to reach all cells of the body. In this respect they hold an important advantage over many other vectors. Table 1 contrasts a number of gene delivery agents with Sindbis and shows some of the distinctive advantages of vectors made from this virus.
TABLE 1
Copending, commonly assigned application Ser. No. 60/279,051, filed Mar. 27, 2001 discloses in vivo studies showing that Sindbis vectors can be used to treat experimental tumors growing in animals and that such treatments can cause substantial necrosis and, in at least one model system, complete elimination of rapidly growing tumors. In addition, it has been shown that Sindbis vectors can be made to target specific cells in vivo and in vitro. Such targeting can be extremely efficient, achieving expression in vitro of a delivered gene in greater than 90% of the targeted cells (35). A number of other laboratories have shown utility for these vectors for the treatment of various conditions (24-27).
Application of viral-based vectors for gene therapy requires efficient processes for their large-scale manufacture. A major obstacle preventing widespread use of Sindbis vectors for gene therapy has been the inability to produce these vectors easily from packaging cell lines. To understand the issues involved in the case of Sindbis vectors, it is important to analyze how these vectors are produced.
Generally, recombinant Sindbis virus is currently produced by the electroporation of two RNA genomes that are synthesized in vitro. One genome, the replicon, directs the synthesis of the gene of interest along with the Sindbis RNA replicase (FIG. 1). This genome also contains a packaging signal allowing it to be incorporated into virion particles by the Sindbis structural proteins. The second, a helper genome (or for greater safety two split structural helper genes; FIG. 2), direct(s) the synthesis of viral structural proteins but lack(s) a packaging signal so that recombinant viral particles will undergo only one round of infection.
FIG. 1 shows the packaging of replication defective replicons by cotransfection of defective-helper RNAs (DHRNAs) expressing the alphavirus structural proteins and the replicon. DHRNAs are designed to contain the cis-acting sequences required for replication as well as the subgenomic RNA promoter driving expression of the structural genes. Packaging of SIN replicons is achieved by efficient cotransfection of cells with both RNAs by electroporation. Replicase/transcriptase functions supplied by the vector RNA lead not only to its own amplification but also act in trans to allow replication and transcription of helper RNA. This results in synthesis of structural proteins that can package the replicon with greater than 107 infectious particles per ml being produced after only 48 h.
In addition to the need to prepare RNA in vitro and electroporate this RNA into cells, the lethality of Sindbis virus requires that this procedure be done anew each time virus is to be produced. In mammalian cells, apoptosis caused by Sindbis virus results in lethality to the infected cells, making it impossible to propagate Sindbis virus infected cells for long periods. To overcome the lethality issue one could construct an inducible system, which would allow propagation of the cells into high numbers before virus production is induced. While the producing cells would still die within days after induction, with an inducible system it would be possible to make large amounts of virus (such as would be needed for clinical applications). By simply preparing large amounts of uninduced cells and freezing aliquots, one could generate a master cell bank from which aliquots could be defrosted as needed, grown to large numbers, and induced to produce virus.
FIG. 2 shows the cotransfection of two DHRNAs along with the expression replicon. One DHRNA (helper 1) expresses the capsid protein and a second DHRNA (helper 2) is designed for high level expression of the virion glycoproteins. Helper 2 uses a deleted version of the capsid-protein that is still able to function as a translational enhancer and an autoprotease but is defective for packaging. This approach is favored because it greatly reduces the possibility that replication competent viruses will arise by recombination. Figure shown is adapted from reference 36.
Polo et al. (37) have developed a “stable” alphavirus packaging cell line using a modification of the two helper system that partially but not fully resolves the need to transfect RNA for vector production. Polo et al. (37) describe the use of an inducible cell system which stably encodes DNA for production of the viral structural proteins, but not the replicon and gene of interest. In this system, translation of the structural proteins (from helpers 1 and 2) is obtained only after synthesis of an authentic subgenomic mRNA by the vector-encoded replicase proteins (which are not encoded by the cells and must be electroporated).
Because the replicon is not present in the packaging cells, to obtain vector production, the Polo et al. (37) system requires electroporation of all the cells with RNA encoding the Sindbis replicon and desired gene. To a large extent this requirement imposes an unnecessary burden in the production of recombinant Sindbis vector for therapeutic application. In particular, given the instability of RNA molecules, large-scale production of in vitro Sindbis RNA genomes is difficult and costly. Further, a complete master cell bank cannot be generated. As Polo et al. (37) must have been aware of this drawback, it is assumed that Polo et al. (37) were not able to design their system in such a way that they could prevent the expression of replicon genes. A reason for not being able to stably integrate the DNA encoding the Sindbis replicon is that production of genes encoded in the replicon, such as nsP2, may be very toxic to mammalian cells.
Therefore, what is needed in the art are packaging cell lines for the production is Sindbis viral vectors which overcome the deficiencies.